Withdrawal from ethanol dependence has been associated with heightened anxiety and reduced expression of Brain-derived neurotropic factor which promotes the synaptic transmission and plasticity of synapses. Hyperpolarization-activated cyclic nucleotide-gated channel 1 regulates expression; however, whether Hyperpolarization-activated cyclic nucleotide-gated channel 1-related Brain-derived neurotropic factor is involved in the synaptic ultrastructure that generates withdrawal-anxiety has been poorly perceived. Sprague–Dawley rats were treated with ethanol 3–9% (v/v) for a period of 21 days. Conditioned place preference and body weight were investigated during ethanol administration. Rats were subjected to behavioral testing and biochemical assessments after ethanol withdrawal, which was induced by abrupt discontinuation of the treatment. The results showed that the ethanol administration induced severe ethanol dependence behaviors, with higher body weight and more time in the ethanol-paired compartment. After withdrawal, rats had a higher total ethanol withdrawal score and explored less. Additionally, increased Hyperpolarization-activated cyclic nucleotide-gated channel 1 protein and gene expression and decreased Brain-derived neurotropic factor protein and gene expression were detected in the Ethanol group. Eventually, there was a negative correlation between the level of Brain-derived neurotropic factor mRNA and Hyperpolarization-activated cyclic nucleotide-gated channel 1 protein. Importantly, the synaptic ultrastructure changed in the Ethanol group, including increased synaptic cleft width and reduction in postsynaptic density thickness or synaptic curvature. The synthesis of the Brain-derived neurotropic factor mRNA could be down-regulated by higher Hyperpolarization-activated cyclic nucleotide-gated channel 1 protein expression. Changes in synaptic ultrastructure may be induced by lower Brain-derived neurotropic factor protein, which could be associated with the withdrawal-anxiety that is experiences after ethanol dependence. Alcohol dependence is chronic relapsing disorder characterized by repeated episodes of withdrawal and then relapse with resumption of heavy alcohol consumption . Abrupt Previous studies found that changes in the synaptic ultrastructure and morphology in the nucleus accumbens (NAc) and hippocampus (Hip) are involved in various behavioral sequelae, including movement control, motivation, and addiction , 5. EthaBrain-derived neurotrophic factor (BDNF), a neuromodulator in the mammalian nervous system, seems to contribute to the neuroadaptive changes set in motion by drug withdrawal . AbstineIt has been found that BDNF could be up- regulated by a lentivirus-based silencing RNA system using short hairpin RNA (shRNA)-hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1), which reduced the expression of HCN1 protein, increased the neuronal excitability, and produced anxiety- and depressive-like behavior . Studiesin vitro study reported that withdrawal from repeated methamphetamine administration increased HCN1 mRNA levels  and were approved by the WeiFang Medical University Animal Care and Use Committee.The male Sprague–Dawley rats  utilized in this study were purchased from the animal center at WeiFang Medical University. The animals were housed five per cage (53.5 × 39 × 20 cm) with a 12-h light/12-h dark cycle (lights on at 06:00 a.m.) at a relatively constant room temperature (23 ± 1°C) and humidity (45%). Food was available n = 20) animals received water ad libitum for 23 days. The ethanol group (n = 20) received ethanol starting with a solution of 3% ethanol (v/v) that was gradually increased every 3 days to 6% (days 7–9) and then 9% (days 10–24). Then, the ethanol solution (9%) was removed and returned the next day (day 24) for 2 h. After that, the animals received water until day 26, thereby ensuring a 48 h abstinence period. Previous studies showed that anxiety-like behavior after this period of abstinence from ethanol was robust  testing with a two-compartment CPP apparatus . In brieTo identify any pre-existing bias toward the individual compartments, each rat was initially placed in the middle chamber and allowed to freely explore the entire apparatus for a 15-min session for three consecutive days before ethanol administration. The time spent in each compartment on the third day was used as the pre-conditioning data. The most preferred compartment was designated as the preferred compartment, and the other as the non-preference compartment. Each animal received individual CPP training. In the post-CPP session, each rat was allowed to freely explore the entire apparatus for a 15-min session repeatedly on Day 22–24 in an ethanol-free state. The amount of time spent in the non-preference (ethanol-paired) compartment was recorded as the post-conditioning data.The symptoms of ethanol withdrawal were tested for 4 min 4 and 6 h after ethanol withdrawal. At each observation time, the rats were simultaneously assessed for the following behavioral conditions: stereotyped behaviors , agitation, tail stiffness, abnormal posturing, and abnormal gait. These behaviors were measured in the open-field test (OFT) (40 × 40 × 35 cm). Before the start of the experiment, the rats were put in the open field arena for 30 s. The symptoms of ethanol withdrawal were scored using a rating scale prepared by Erden . The behExploratory locomotor activity and anxiety were tested in the OFT as previously reported . The areThe standard EPM test was used to assess anxiety-like behavior . The EPM2O2 in methanol. After rinsing in phosphate-buffered saline (PBS) three times, the sections were blocked in 5% normal goat serum at room temperature. Next, the brain sections were incubated overnight at 4°C in primary antibody  solutions with Primary Antibody Dilution Buffer. After repeated washing, the brain sections were incubated at room temperature with secondary antibody (Peroxidase-Conjugated Affinipure Goat Anti-mouse IgG (H+L), 1:200 dilution, ZB-2305, ZSGB-BIO, 1:200 dilution; Peroxidase-Conjugated Affinipure Goat Anti-Rabbit IgG (H+L), 1:200 dilution, ZB-2301; ZSGB-BIO, Beijing, China) and reacted with a 3,3′-diaminobenzidine (DAB) kit  for the color reaction. The Hip and NAc sections were examined with an Olympus Fluo View 1200 confocal microscope system , and photomicrographs of representative Hip and NAc areas were obtained.Immunohistochemistry was performed as previously described . The ratThe western blot assay was conducted as previously described . The Hip−ΔΔCT method with β-actin as an endogenous control.The rats were euthanized after treatment, and the Hip and NAc were extracted from each group and analyzed. Total RNA was isolated using TRIzol reagent . The RNA concentration and purity (OD260/280) were determined using a NanoDrop ND1000 spectrophotometer . First-strand cDNA synthesis was performed using the RevertAid™ First Strand cDNA Synthesis Kit  in a 20-μL reaction volume with 1 μg template RNA. The PCR reaction mix was prepared according to the manufacturer's protocol. The PCR primer sequences are shown in Table The TEM test was carried out as described by Marcelo . The HipU-test. Between-group differences in body weight were analyzed using repeated-measures analysis of variance (ANOVA). Other data are expressed as the mean ± standard error. Student's t-test was used to compare the differences between the control and ethanol groups using SPSS 22.0 software . The correlation between the HCN1 protein and BDNF mRNA as well as the BDNF protein was tested by correlation analysis. A P < 0.05 was considered significant.All measurements were obtained by an independent investigator blinded to the experimental conditions. Total ethanol withdrawal scores in the different groups were compared using the Mann–Whitney F = 25819.874, P < 0.001; Figure The rats in the ethanol group gained significantly more weight than rats in the Control group  as well as the NAc . However, the level of the HCN1 protein had no correlation with the BDNF protein in the Hip  or the NAc .Because BDNF and HCN1 play a key role in ethanol withdrawal-induced anxiety, we performed a western blot analysis of BDNF and HCN1 in the Hip and NAc. As shown in Figures t = 9.776, P < 0.001, Figures t = 6.169, P < 0.001; Figures t = 5.225, P < 0.001; Figures t = 14.618, P < 0.001; Figures The immunohistochemistry results of the ethanol group showed significantly fewer BDNF-positive cells in the Hip  that reduces HCN1 channel function leading to an increase in BDNF synthesis is unknown .The change in Ih in the HCN1 channel caused by ethanol withdrawal is mediated by increases in cAMP, which binds to a cyclic nucleotide binding domain (CNBD) on the COOH-terminus of the channel. The HCN1 channels are activated by the CNBD . HoweverDespite the important role played by Hip and NAc in mediating ethanol withdrawal-induced anxiety, we were unable to ascertain whether or not there is any interaction between Hip and NAc. Additionally, Neuropeptide Y (NPY) is a neuromodulator that is involved in the regulation of alcohol dependence and withdrawal, as well as the BDNF , 50. BotIn summary, we provide several lines of new evidence for the increase in anxiety-like behaviors exhibited by rats undergoing ethanol withdrawal that possibly resulted from abnormal changes in synaptic ultrastructure. These findings speculate that the extended synaptic ultrastructure might be regulated by HCN1-related BDNF mRNA expression during ethanol withdrawal-induced anxiety, which might lead to a better understanding of the synaptic systems affected by ethanol withdrawal and provide new perspectives for the development of appropriate therapies for ethanol withdrawal-induced anxiety.LH, LS, and YG designed the study. LH and YG collected the data. LH analyzed data and drafted the manuscript. BL, YW, CL, GW, QL, JP, HS, and LS reviewed the manuscript. All authors contributed to and have approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Alzheimer's disease (AD) is a slowly progressive disease characterized by impairment of memory and eventually by disturbances in reasoning, planning, language, and perception. Ageing is the greatest risk factor for its development but mutations in amyloid precursor protein (APP), apolipoprotein E (APOE), microtubule-associated protein tau (MAPT) among others, are also a major factor  is a model that naturally integrates multiple AD pathological hallmarks like tau fibrilary tangles and β-amyloid deposits  were captured from a natural population in central Chile, 30 km west of Santiago . In this environment, degus typically breed once per year in late autumn (May-June), with conceptions in late winter to early spring (September-October). The animals were captured as juveniles during early austral summer (November), when degus are 2–3 months old, with mesh made traps with a Sherman-trap type mechanism. Juveniles corresponded to individuals weighing 70–130 g  or 70–140 g , and hence it is possible to differentiate them categorically from adults, which weight above 130 g for females, 140 g for males  of 3-years old O. degus was performed using the burrowing test in an earlier work , and three samples from GB animals (with soluble levels of Aβ1−42 used as control group) were used for further analysis. Brain tissues were frozen directly in RNAlater solution (Ambion) and stored at −80°C until use. All procedures of capture, transportation, maintenance and experimentation followed the recommendations of the ethics committee of the Faculty of Sciences of the University of Chile, and complied with Chilean regulations (SAG-Chilean Agriculture and Livestock Service) as well as recommendations by the Animal Behavior Society.They were housed in standard metal cages 50 × 40 × 35 cm with a layer of wood shaving as bedding and containing a small metallic box (25 × 15 × 10 cm with a single entrance), under natural photoperiod  and in an air-conditioned animal facility at the Faculty of Sciences, University of Chile . They were fed with a commercial rodent diet . Water and food were provided For total RNA extraction, PureLinkTM RNA Mini kit  was used according to the manufacturer's instructions. For genomic DNA digestion, RNase-Free DNase I (Ambion) was used. Total RNA samples were quantified in a Nanodrop 2000 spectrophotometer and integrity was evaluated by agarose gels electrophoresis with a standard protocol. For mRNA isolation from total RNA samples, MicroPoly(A) Purist kit was used according to the manufacturer's instructions. For sizing, quantitation and quality control of mRNA Bioanalyzer system (Agilent) was used, discarding samples with an RIN < 7.RNA-seq was performed at the National Center for Genomics and Bioinformatics  including library preparation , fragmentation and PCR enrichment of target mRNA according to strand-specific protocols. Two batches of paired-end (75 × 35 bp) library sequencing were done in a SOLiD 5,500 × l system (Applied Biosystems).ab initio and evidence-based approaches by using the Program to Assemble Spliced Alignments (PASA) and Evidence Modeler (EVM) to de novo annotate protein coding genes in O. degus genome assembly (v1.0)  1,741,759 ESTs and mRNA sequences from four species of rodents: mouse, guinea pig, Chinese hamster and rat. Transcript sequences for all five species were derived from the GenBank NCBI databases. We also used >30 million UniRef90 protein clusters and 26,259 highly curated rodent-specific Swiss-Prot proteins that were split-mapped to the O. degus genome by using SPALN2  and ab initio gene models were combined into consensus CDS models using Evidence Modeler  Haas, . In totans Table . We alsoO. degus genome were further functionally annotated using an in-house developed, automatic pipeline. For each protein sequence we assigned protein signatures, orthology groups, as well as annotated metabolic pathways and reactions using an orthology-based approach. In this analysis we used InterProScan v.5  to the predicted genes. This was done by taking NCBI gene names from the corresponding, NCBI annotated proteins, showing full sequence similarity. The sequence similarity was measured by assigning SHA1 checksum to each protein in both proteomes, followed by comparing those sums.Predicted protein-coding genes in the O. degus RNA Sequences were aligned to a reference transcriptome obtained from the masked primary genome assembly version 1.0 (WGS Project: AJSA01) and the EVM-based genome annotation. The transcriptome sequence was prepared using RSEM version 1.2.12 . Next, pair-end reads were mapped to the human reference transcriptome using our in-house pipeline that combines STARv.2.4.0.1  was derived from the National Center for Biotechnology Information Sequence Read Archive database  controls, the absolute value of log2 Ratio (logFC ≥ 1), log read count per million reads (logCPM > 1) and FDR < 0.05 were used as a criterion. Raw gene reads counts were also normalized to RPKM values (reads per kilobase per million mapped reads) using the RPKM formula described by Mortazavi et al.  based annotation of repeat-mask O. degus genome (v1.0) resulted in prediction of 31,739 protein-coding genes, corresponding to 36,866 predicted transcripts and 36,575 proteins. The comparison of EVM-based and NCBI reference annotation revealed that the number of protein coding genes increased by 1.52-fold from 20,779 (NCBI) to 31,739 (EVM-based). Similar 1.40-fold change was reported for protein coding transcripts  (Table With the aim to enrich the current version of n) Table .Each protein sequence was functionally annotated using the in house automated pipeline. Annotation features were assigned to a total of 35,618 (97%) proteins, 30,661 (97%) genes), of these 29,847 (82%) of the proteins were assigned some GO terms Tables , S2. ThiO. degus genome, we performed pairwise comparisons to measure the gene expression level differences between AD-like subjects and controls. As a result 54 DEGs were identified between those two groups. Statistical analysis revealed 29 genes to be up- and 25 to be down-regulated in our study  in mitochondria. This is consistent with previously published data, as AD has been already linked to low activity of brain cytochrome c oxidase  enrichment analysis was performed Table . The up-O. degus could be employed as a natural model for studying the pathogenesis of AD, a human AD brain transcriptome was analyzed using RNA-seq data derived from hippocampus tissue of AD subjects and controls  this included COX16, APOC1, ITGBL1, FGF9, and AMD1. All five DEGs correlated with MMSE scores, while three: ITGBL1, FGF9, and AMD1 were both MMSE and NFT-correlated. However, only AMD1 showed a similar correlation with incipient AD . Our results revealed in O. degus a number of genes previously implicated in AD and related disorders indicating that this model is a suitable alternative to transgenic mice models for AD research. Because the AD-pathology can vary widely with animal age we carefully matched control and AD-like groups. Animals aged 3 years have optimal AD-like pathology development namely, the presence of beta-amyloid deposits as has been previously reported   that has been associated with the reduction of neuronal energy metabolism and mitochondrial dysfunction observed in AD patients by the inhibition of COX activity as a result of binding to Aβ-subunits , WISP1 and Copine VII (CPNE7). Interestingly, the FGF9 gene is known to be related to the MAPK pathway affected in AD patients  to be deregulated in AD-like O. degus brain samples. Notably, APOC-I is part of the APOE/C-I/C-IV/C-II gene cluster on the chromosome 19 , Presenilin 1 (Psen1), and Aβ-peptide sequences in correlation with specific human variants associated with AD. Their findings revealed that O. degus have the arginine substitution present in the ApoE4 pathogenic human allele; Psen1 gene showed a greater relatedness between the isoforms of human, degus, guinea pig, and mole rat, compared to those of others rodents such the rat, and also described that O. degus Aβ-peptide sequence presents high homology with the human protein, differing in only one amino acid (Salazar et al., O. degus have high levels of Aβ-peptide aggregates, APP, TNF-a, IL-6, IFN-a, and the oxidative stress marker NFE2L2 when compared to healthy control O. degus, suggesting that AD-like O. degus present increased inflammation as is observed in human AD patients (Deacon et al., O. degus shares common affected genes with those have been implicated in human AD and therefore provides further characterization of the O. degus as a relevant natural model. O. degus shows a correlation of expression with human AD-related genes making this model a powerful preclinical tool to characterize target effects of novel therapeutics in AD. This study provides strong evidence to support the O. degus as a valuable natural model for preclinical work in AD research.In this study we focused in whole brain, so studies of additional brain regions are needed and may point to different genes and additional pathways. The RNA-seq uncovered multiple DE transcripts, several of these are novel and some have been previously implicated in AD like WISP1 and APOC1. Being the present work a pilot study we have probably uncovered only a fraction of what these data may reveal about this model and its link with AD supporting further work with this species. Future work is underway in our laboratory to elucidate the characterization of global gene expression of young and old r et al. , compareThis study was carried out under the approval of the ethics committee of the Faculty of Sciences, Universidad de Chile, directed by Dr. Marco Méndez and integrated by:-Dr. Eduardo Friedman-Dr. Victoria Guixé-Dr. Madeleine Lamborot-Dr. Roberto Morales-Dr. Aurelio San Martín-Dr. Cecilia Vergara.Octodon degus transcriptome data is available through the NCBI using accession PRJNA326273.All the FA and BU contributed to the experimental procedures, data analysis and the manuscript preparation. FC, AV, EP, SN, and DL contributed to created the bioinformatic pipeline for sequencing data analysis. LF contributed to the experimental procedures and data analysis. RMJD, MH, and RD contributed to comprehensive analysis of the results and to the manuscript preparation. RV, RG, and PC contributed to the funding of the project, the experimental design and the manuscript preparation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


I-V characteristics reveals that the higher the temperature, the lower the current. This suggests that no single-level traps, but only multiple-level traps, exist in the amorphous ZnO:B films. Accordingly, the trap energy levels (0.46–0.64 eV) and trap spacing (1.1 nm) in these multiple-level traps are extracted.This paper addresses the trap exploration in amorphous boron-doped ZnO (ZnO:B) films using an asymmetric structure of metal-oxide-metal. In this work, the structure of Ni/ZnO:B/TaN is adopted and the ZnO:B film is deposited by RF magnetron sputtering. The as-deposited ZnO:B film is amorphous and becomes polycrystalline when annealing temperature is above 500 °C. According to the analysis of conduction mechanism in the as-deposited ZnO:B devices, Ohmic conduction is obtained at positive bias voltage because of the Ohmic contact at the TaN/ZnO:B interface. Meanwhile, hopping conduction is obtained at negative bias voltage due to the defective traps in ZnO:B in which the trap energy level is lower than the energy barrier at the Ni/ZnO:B interface. In the hopping conduction, the temperature dependence of  Namely, bandgap energy of 2.99 eV  can be achieved by doping with Cd2+, while Mg2+ increases the bandgap energy to 3.9 eV  [Zinc oxide (ZnO) is an attractive material for the applications of semiconductor devices . It has  = 0.33) ,6,7. ZnO = 0.33) .2) or indium tin oxide (ITO) as a transparent conducting electrode in liquid crystal displays or solar cell devices due to the advantages of abundant raw material, low synthetic temperature, available large single crystal, amenable wet chemical etching, simple manufacturing process, competitive optical and electrical properties, nontoxic and stable in plasma, and radiation hardness [In general, ZnO with a wurtzite structure is an unintentional n-type semiconductor because of the deviation from stoichiometry. The background free electrons basically result from the shallow donor levels related to the presence of native defects such as oxygen vacancies and/or zinc interstitials . To achihardness ,9. Note hardness . Recentlhardness ,4. In adhardness .I-V) characteristics, Ohmic conduction is obtained at positive bias voltage due to the low work-function electrode TaN which forms Ohmic contact at the TaN/ZnO:B interface. Whereas, hopping conduction dominates at negative bias voltage due to the defective traps in ZnO:B in which the trap energy level is lower than the energy barrier at the Ni/ZnO:B interface. Lower energy obstacle leads to higher carrier transport, and therefore dominates the conduction current through the oxide. In the hopping conduction, the temperature dependent I-V characteristics reveal that the higher temperature, the lower current. This implies that the current decrease at higher temperature results from the multiple-level traps existed in the amorphous ZnO:B films.Because the defect issue is generally critical in the ZnO:B-based devices, the carrier trapping characteristics in ZnO:B films is important. For thermal budget reduction, the as-deposited amorphous ZnO:B films grown by RF magnetron sputtering are concerned. In this work, the trap exploration in the amorphous boron-doped ZnO films was studied. An asymmetric metal-oxide-metal (MOM) structure with ZnO:B was fabricated and investigated for the studies of metal/oxide interface property and defect trap nature in ZnO:B films. The structure of Ni/ZnO:B/TaN was used. Based on the analysis of current-voltage . According to the experimental results, carbon contamination level was extremely low in our films deposited from the ZnO:B source material and was below the detection limit of the Auger electron spectroscopy system . c-axis. The mean grain size of both 500 °C- and 600 °C-annealed ZnO:B films estimated by Scherrer formula according to the full-width at half-maximum (FWHM) of (002) peak in To examine the microstructure of boron-doped ZnO (ZnO:B) films, the X-ray diffraction (XRD) patterns were measured at room temperature using a powder diffractometer  and electric field (E) can be adopted [q is the electronic charge; a is the hopping distance ; n is the electron concentration in the conduction band; v is the frequency of thermal vibration of electrons at trap sites; E is the applied electric field; T is the absolute temperature; k is the Boltzmann’s constant; and CE). In this work, the electron concentration is about 1018 cm−3 in the ZnO:B films according to the Hall measurement. Based on Equation (1), the mean trap spacing can be determined by the slope of the linear part of log(J) versus E at each temperature. Hence, the trap spacing in ZnO:B films is extracted to be 1.1 ± 0.1 nm according to qaE and the trap energy level i.e., breakdown field 1.5 MV/cm) and average hopping distance (1.1 nm), the maximum field energy 18 cm−3 in the ZnO:B films according to the Hall measurement. Moreover, the frequency of thermal vibration of electrons at trap sites can be qualitatively represented by the frequency of optical phonons in the solid [−1 [13 Hz. In this work, the frequency of 1 × 1013 Hz was assumed. The deviation induced from the uncertainty of optical phonon frequency in ZnO:B films is smaller than 0.02 eV. This value in the determination of trap energy level can be neglected in this work. Hence, the temperature dependence of trap energy levels in ZnO:B films were obtained, as shown in i), singly charged zinc interstitials (Zni+), and doubly charged zinc vacancies (VZn2−). Zni, Zni+, and VZn2− are all the defects of non-lattice ions in ZnO films. The defect energy levels of Zni, Zni+, and VZn2− in ZnO films are 0.46, 0.5, and 0.56 eV, respectively [i, Zni+, and VZn2− may play the important roles in the current conduction in ZnO:B films. These defects may be introduced during the ZnO:B deposition process. Consequently, not single-level but multiple-level traps were found in the amorphous ZnO:B films. Note that defects, such as interstitials and vacancies, are imperfections in the crystal lattice. Interstitials signify extra atoms occupying interstices in the lattice. Meanwhile, vacancies signify missing atoms at regular lattice positions. In addition, current flow through the oxides will be raised at higher temperature for oxide films with the single-level traps. Based on our previous study [Since metal-oxide interface plays an important role in current conduction in a metal-oxide-metal structure, different metal electrodes are adopted to investigate the carrier transportation in this work. Nickel (Ni) is a high work-function metal and its value is 5.15 eV . Whereasent ways . General adopted . The sim adopted :(1)J=qahe solid . The phoectively . This reus study , the sinEC). To explore the chemical defects in the amorphous ZnO:B films, the X-ray photoelectron spectroscopy (XPS) spectra were used to examine the chemical states of zinc and oxygen. Thermo Fisher Sceientific Theta Probe XPS system  was used to collect the photoelectron spectra of the samples with a take-off angle of 90° relative to the sample surface. The vacuum pressure was below 10−9 torr during spectra data acquisition and using high resolution scans (0.02%~2%). In order to obtain meaningful binding energies, charge referencing was performed for the XPS measurements. In the beginning of the XPS measurements, the binding energy of the photoelectron was calibrated by assigning 284.8 eV to the C1s peak corresponding to adventitious carbon. For detecting the binding energies in the middle part of ZnO:B films, XPS spectra were collected after sputter-cleaning with 1-keV Ar+ ions for 1.2 min. 3+ in B2O3 structure, which provides the evidence for the incorporation of boron into the zinc oxide [1/2 and 2p3/2 for Zn2+ correspond to the peaks at 1044.7 and 1021.5 eV, respectively [1/2 and 2p3/2 for nonlattice zinc ions are located at 1043.9 and 1021.1 eV, respectively. According to 1/2 and 2p3/2) of nonlattice zinc ions is much more obvious than that (O 1s) of nonlattice oxygen ions. Namely, the peak area ratio of nonlattice to lattice zinc ions is much higher than that of nonlattice to lattice oxygen ions. This implies that the number of zinc deficient states is much larger than that of oxygen deficient states. As a consequence, the defects related to nonlattice zinc ions play the more important role in the current conduction in the amorphous ZnO:B films. A literature report pointed out some defect energy levels regarding nonlattice zinc and oxygen ions [i), singly charged zinc interstitials (Zni+), and doubly charged zinc vacancies (VZn2−). The defects related to nonlattice oxygen ions include neutral oxygen vacancies (Vo), singly charged oxygen vacancies (Vo+), and oxygen interstitials (Oi). The defect energy levels of Zni, Zni+, and VZn2− are 0.46, 0.5, and 0.56 eV, respectively. Furthermore, the defect energy levels of Vo, Vo+, and Oi are 1.62, 2, and 2.28 (or 2.96) eV, respectively. According to the simulation work on the hopping conduction current in ZnO:B films, the trapping level of the multiple-level traps is about in the range of 0.46–0.64 eV at 25–100 °C. This result also suggests that the defects related to the nonlattice zinc ions play the key role in the current conduction in the amorphous ZnO:B films. Note that the location of oxygen vacancy state can correspond to the activation energy obtained in the experiments. Aside from ZnO films in this work, the activation energy values in various dielectric materials can be found in the literature. One of the references indicated that the activation energy values of the different oxygen defects are in the range of 0.1–0.5 eV for the singly ionized oxygen vacancies, 0.6–1.2 eV for the doubly ionized oxygen vacancies, and 0.9–1.1 eV for the diffusion oxygen vacancies [According to the study of current conduction mechanisms in this work, we revealed that the trap energy levels in ZnO:B films are around in the range of 0.46–0.64 eV below the conduction band edge  magnetron sputtering in argon ambient at room temperature using a ceramic ZnO:B target. The boron doping concentration of ZnO:B films was about 0.8 wt %. The flow rate of argon was 20 standard cubic centimeters per minute (sccm). The working pressure during deposition was 4 mTorr. The RF power was 60 W. The deposited ZnO:B film thickness was 27 nm. To investigate the crystal properties of ZnO:B films, rapid thermal annealing (RTA) was performed in N2 for 30 s at temperatures ranging from 400 °C to 600 °C. To achieve the MOM structure, a nickel (Ni) top electrode was deposited by thermal evaporation with a round area of 3.14 × 10−4 cm2 patterned by the metal shadow mask. The electrical characteristics of the fabricated Ni/ZnO:B/TaN capacitors were measured by an semiconductor parameter analyzer . During the measurement, the voltage bias was applied on the top electrode (Ni) with the bottom electrode (TaN) grounded. All the measurements were performed under dark condition.In this work, the metal-oxide-metal (MOM) capacitors were fabricated. The boron-doped ZnO (ZnO:B) thin films were deposited on TaN/SiOIn conclusion, the trap properties in amorphous boron-doped ZnO films were studied using the structure of Ni/ZnO:B/TaN. The Ohmic conduction dominates at positive bias voltage; meanwhile, the hopping conduction dominates at negative bias voltage. Based on the analysis of current conduction mechanism, we revealed that the defects with multiple trap levels exist in the amorphous ZnO:B films. These defects are related to the nonlattice zinc ions and play the key role in the current conduction. Simulation results show that the defect trap spacing between the defect sites is around 1.1 nm. Furthermore, the defect trap level increases with increasing temperature.


During and after each heat stress (HS) treatment, temperature were monitored for air, canopy, and soil; net CO2 assimilation (Anet), quantum yield of photosystem II (ΦPSII), stomatal conductance (gs), and internal CO2 level (Ci), specific leaf area (SLA), and chlorophyll content of the dominant species were measured. One week after the last HS treatment, all plots were harvested and the biomass of above-ground tissue and flower weight of the two dominant species were determined. HS decreased physiological performance and growth for both species, with S. canadensis being affected more than A. gerardii, indicated by negative HS effect on both physiological and growth responses for S. canadensis. There were significant timing effect of HS on the two species, with greater reductions in the net photosynthetic rate and productivity occurred when HS was applied at later-growing season. The reduction in aboveground productivity in S. canadensis but not A. gerardii could have important implications for plant community structure by increasing the competitive advantage of A. gerardii in this grassland. The present experiment showed that HS, though ephemeral, may promote long-term effects on plant community structure, vegetation dynamics, biodiversity, and ecosystem functioning of terrestrial biomes when more frequent and severe HS occur in the future.Heat-waves with higher intensity and frequency and longer durations are expected in the future due to global warming, which could have dramatic impacts in agriculture, economy and ecology. This field study examined how plant responded to heat-stress (HS) treatment at different timing in naturally occurring vegetation. HS treatment (5 days at 40.5°C) were applied to 12 1 m Howeverd growth . To dated growth  or arctid growth , 2006. IThe negative effects of HS on plants growth and crop yield mainly was caused through its negative effects on photosynthetic process, which is among the most thermosensitive aspects of plant functions . Due to 4 species than for C3 species because C4 species usually have higher water use efficiency and lower photorespiration due to its CO2-accumulating mechanisms in the leaf  to determine how HS affects the ecophysiological and morphological characteristics of a C4 and C3 species which co-dominate a restored prairie community; (2) to determine the effect of timing of HS on each species growth and physiology. Our specific hypotheses were as follows: (1) HS will have a less pronounced negative effect on the C4 than the C3 species; (2) differences in the responses to HS applied at different growing stage (HS timing effect) will lead to differences in plants ecophysiological responses and growth.To examine the influence of HS on plants ecophysiological and growth response in naturally occurring mixed C1. Andropogon gerardii (big bluestem), a warm-season C4 perennial grass, and Solidago canadensis (goldenrod), a warm-season C3 perennial herbaceous dicot, together account for almost 95% plant canopy cover and the majority of total aboveground productivity in this ecosystem. Top-vented 1 m3-chambers made with transparent plastic attached to a wooden frame was used to simulate HS treatment. Heat treatment was applied in situ from June 21 to June 25, July 22 to July 26, and August 28 to September 1 in 2007 , within the oak-savannah glacial-sand ecosystem referred to as “Oak Openings” region07 as in . There w07 as in . Heat-tr2 level were measured daily with a portable infrared gas analyzer . During measurements, CO2 concentration of 380 μmol mol-1, leaf temperature of 25°C, photosynthetic photon flux (PPFD) of 1500 μmol m-2 s-1 and airflow through the chamber of 250 μmol s-1 were set in the leaf chamber. Net photosynthetic rate (Anet) was taken as the rate of photosynthesis at a PPFD of 1500 μmol m-2 s-1. The parameters including stomatal conductance (gs) and intercellular CO2 concentration (Ci) were recorded during the photosynthetic measurement. Intrinsic water use efficiency (iWUE) was calculated as the ratio of net photosynthetic rate to stomatal conductance. Quantum yield of PSII electron transport (ΦPSII) was measured with a pulse-amplitude-modulated (PAM) fluorometer with a saturating pulse of 3000 μmol photons m-2 s-1  on ambient light-adapted (∼800 μmol photons m-2 s-1) plants, as in 2 leaf punches from each leaf were taken and oven-dried at 65°C for 2 weeks for measurement of SLA (m2 kg-1) and LWC (%). An index of the total leaf chlorophyll content was measured using a chlorophyll meter . Readings were taken along the middle section of the four leaves of one plant and the mean value was used for analysis. The measurements were made on five plants from each treatment before, during and after the HS.Photosynthetic measurements were conducted during and after each HS treatment in order to determine the timing effect of HS on foliar gas exchange. During and after each heat treatment, one fully expanded leaves were chosen randomly from each plot and net photosynthetic rate, stomatal conductance to water vapor, and internal COFour-week after the last HS treatment, 40 cm × 50 cm of each plot was harvested. The clipped plants were sorted into different categories , oven-dried at 65°C for 1 week and weighed.3. The normality of the residuals of all the variables was tested using the Shapiro–Wilk test. Fixed effects of species, heating stress at different time and their interactions on the morphological, biochemical, and physiological parameters were tested by a linear mixed-effects model, using the lme4 package4. The measuring time were specified as a random factor to control for their associated intra-class correlation. Linear mixed-effects models also tolerate the necessarily unequal number of responses and unbalanced sample sizes for each treatment. We obtained p-values for regression coefficients using the nlme package. For the sake of brevity, we present only the F tests from the LMER results here . A Post hoc Tukey HSD tests were made on specific contrasts to examine significant treatment effects among groups . End of season measurements of aboveground primary production, flower weight and leaf morphological parameters were analyzed via t-tests to account for heat-stress timing effect. All statistical tests were considered significant at P ≤ 0.05. Mean values of each variable were expressed with their standard error (SE).All statistics were tested in the R statistical languageA. gerardii and S. canadensis in heated plots was higher than that in control plots, but returned to control levels right after the end of HS . For HS applied during early-, peak-, and reproductive- growing season, leaf temperature reached 34.9, 34.3, and 33.2 for A. gerardii and 34.1, 34.1, and 33.8°C for S. canadensis in the heated plots, respectively .Air temperature in the heated plots increased on average to 40.5 ± 2.8°C during HS treatment . ANPP of the plots heat-stressed at reproductive-growing season was significantly lower than that of the control plots. The productivity of S. canadensis, but not A. gerardii, was significantly reduced by HS. The flower weight of S. canadensis was higher than that of A. gerardii, but neither was affected by HS . LAI was significantly lower at the plots heat-stressed at reproductive growing stage than the control plots . LAI was highest at the plots heat-stressed at early-growing season and lowest at the plots heat-stressed at reproductive-growing stage .Aboveground net primary production at the end of growing season differed significantly among different treatments . SLA of plants heat-stressed at peak- and reproductive- stages was significantly higher than that heat-stressed at early-growing stages. LWC of S. canadensis was higher than that of A. gerardii. Compared with control plots, HS at reproductive-growing stages significantly decreased LWC for S. canadensis. And for A. gerardii, LWC of plants at the plots heat-stressed at peak-growing stage was significantly lower than that at control plots and plots heat-stressed at early-growing stages .Specific leaf area of 2 assimilation rate and stomatal conductance in heat-stressed plants. Anet (net photosynthetic rate) was higher for A. gerardii than that of S. canadensis. Anet was significantly lower in heated plots than in control plots during HS for A. gerardii and S. canadensis . Anet remained depressed for at least 1 week after HS in heated plants, relative to unheated plants. Throughout the experimental duration, Anet was significantly decreased by heat-stress at peak and reproductive growing stages, compared with control plots. Anet was lowest for the plots heat-stressed at reproductive stage, followed by the plots heat-stressed at peak-growing and early-growing stage . Stomatal conductance to water vapor (gs) varied among different species and treatment. For A. gerardii and S. canadensis, gs was lower in heated plots. There was also a similar significant timing effect of HS on gs as on Anet, with lowest gs achieved at plots heat-stressed at reproductive stage . Variation in internal CO2 (Ci) was also a function of species and treatment. For A. gerardii and S. canadensis, Ci was higher in heated plots. There was also a significant timing effect of HS on Ci, with highest Ci achieved at plots heat-stressed at reproductive stage than that of control plots and plots heat-stressed at early- and peak- growing stages . The intrinsic water use efficiency (iWUE) was lower for the plants heat-stressed at the reproductive stages than control plants. There was no significant difference between control plots and plots heat-stressed at early- and peak- growing season .Heat stress treatment reduced net COPSII) was higher for S. canadensis than for A. gerardii. HS at different growing stages played a significant role in affecting ΦPSII. HS decreased ΦPSII significantly when it was applied at the peak-growing season, compared with control plots . The content of chlorophyll (chlorophyll a + chlorophyll b) was significantly different among different treatments. For both species, chlorophyll content in newly developed leaves was significantly lower than that in the fully developed and senescent leaves . The chlorophyll content was affected by heat-stress at different growing stages significantly. HS applied at different growing stages all lowered chlorophyll content significantly compared with control plots for both species. Chlorophyll content of the two species heat stressed at peak and reproductive growing stage was lower than that heat-stressed at early growing stages .Quantum yield of PSII electron transport  the physiology of both species and growth of S. canadensis were affected by HS treatment; (2) the degree of HS effect varied when it applied at different growing stages, with greater negative effect associated with HS applied at later-growing season; (3) the physiology and growth of the two dominant species showed differential sensitivity to HS, with S. canadensis being affected more than A. gerardii.Extreme climate events have long been acknowledged as a universal phenomenon in recent years and caused great agricultural, economic and ecological consequences . HoweverA. gerardii (C4) and S. canadensis (C3) experienced decreased Anet and intrinsic water use efficiency during HS. The decreases in Anet were still evident 1 week after heat treatment ended and the recovery to the control level took at least 1 week, which indicates that under moderate HS conditions (most commonly reported at temperatures between 35 and 40°C), photosynthesis can be reversibly reduced  for both species  measures the proportion of light absorbed by chlorophyll associated with PSII system that is used in photochemistry . The significantly decreased ΦPSII suggested that both species engaged flexible heat dissipation in response to HS, presumably because the activation of Rubisco was inhibited at higher temperatures . However, the C3 species, S. canadensis, tended to close stomata in response to HS, leading to reduced transpiration . In contrast, the C4 species, A. gerardii, tended to keep stomata relatively open and maintained high transpiration rates which would limit negative temperature effects on the foliage.The optimal temperature for photosynthesis ranges between 20 and 35°C for most plant species . Howeverlevation , 2012. Hlevation  or the tlevation . In contS. canadensis. Mid- or late- summer heat event was associated with strong physiological stress . Consistently, the physiological performance of Anet, gs, and ΦPSII were all decreased more during HS applied at peak- or reproductive- growing stages.The response of plants to HS was variable depending on the season or life stage during which the HS event happened. Plants were reported to be more susceptible to HS during later reproductive developmental stages . Early-gl stress . In thisFigure 2B), which is contrary to what have reported that heat-stressed plants decreased flower production and produced later flowers on existing inflorescences (S. canadensis had similar mortality rate, while A. gerardii showed no mortality among different treatments. Eryngium alpinum L. during HS, while adult plants were less affected. Similarly, mortality of the Tenerife endemic Helianthemum juliae seedling reached nearly 100% in years of extreme drought (Notably, in this study, the flower weight of the two species was not affected by HS (escences . HS appl drought .S. canadensis being affected more than A. gerardii, indicated by the negative HS effect on both physiological and growth responses for S. canadensis; secondly, there were significant timing effect of HS on the two species, with greater reductions in photosynthesis and productivity occurred when HS was applied at later-growing season. The reduction in aboveground productivity in S. canadensis but not A. gerardii can increase the competitive advantage of A. gerardii, which therefore could have dramatic implications for species abundance, distribution and community structure. The present experiment showed that ephemeral HS may promote stochastic successions at the community level (3 and C4 plants. Furthermore, this study focused on plants ecophysiological processes and only examined short-term plant responses to HS within one generation of perennial plants. The results suggest that long-term effect of HS on plant communities and ecosystems dynamics should be studied more extensively and with longer experimental durations, particularly in combination with other potentially interactive aspects of global environmental change including increases in atmospheric CO2 and O3 concentration and altered precipitation pattern (Understanding the responses of dominant species to climate extremes is essential to predict future ecosystem dynamics and is particularly important when these species experience similar growing phenology but differ in their sensitivities to the climate factors. In this study, we examined the timing effects of HS on two dominant species in a tallgrass prairie ecosystem. There were two main conclusions drawn from this research. First, the photosynthetic and growth responses of these two species to HS were different, with ty level  or promoty level . It is w pattern ,b.DW and SH came up with the research idea. DW led the experiment and writing. KM and RT assisted conducting the experiment and revising the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's disease in ruminants and there has been a shift in the public health approach to MAP and human diseases like Crohn's disease. The prevention of infection by MAP in ruminants is thought to deter the high impact of economic losses in the level of dairy industry and possible spreading of this pathogen in dairy products. The present study was done to investigate the construction and expression of the soluble form of a novel fusion protein, consisting of Heparin-binding hemagglutinin (HBHA) and high antigenic region of Fibronectin Attachment Protein-P (FAP-P), in order to introduce as a Th1 inducer subunit vaccine against MAP. HBHA is a mycobacterial adhesin and it has been demonstrated that a HBHA-specific IFN-γ response, in latent M. tuberculosis infection, depends on the methylation of the antigen. Further, FAP-P induces Th1 polarization. Because methylation of HBHA was not performed in E. coli, Pichia pastoris was chosen as the host. The desired fusion protein had a similar 3D structure to that of HBHA with its native form and post-translational methylation in C-terminal. Hence, the uptake of the purified fusion protein will be done by M cells because of HBHA, and cell-mediated immunity will be induced because of both antigens. Eventually, successful construction and expression of the newly-designed chimeric protein under the mentioned conditions is reported in this article. Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's disease that infects both domestic and wild ruminant animals. The important effect of this disease is its economic significance in the level of dairy industry [1-mediated immune responses is crucial to reducing the number of bacteria in the early stages of Johne's disease. Next, it is the induced strong Th1 responses which are essential to the development of subunit vaccines [M. tuberculosis has post-translational modification consisting of methylation of lysines in the C-terminal domain [M. tuberculosis infection [1 polarization and IFN-γ production by T cells in vitro [Escherichia coli and Pichia pastoris, methylotrophic yeast with high level of production of recombinant proteins is an important diverse post-translational modification for HBHA, including polypeptide folding and methylation. In addition, growing to high cell densities has become an interesting and important alternative to bacterial expression systems [P. pastoris (strain GS115) expression system is employed in the expression of authentic heterologous proteins of high quality and high quantity to be secreted in a soluble form [ Pichia pastoris as a host because methylation of HBHA was not performed in E. coli, and we were able to confirm secretory expression of a soluble form of chimeric protein with an expected size of 32.9 kDa in culture media using western blot.industry , 2. Therindustry . In addiindustry , type 1 industry , Hashimoindustry , and mulindustry  were stuindustry . Until nindustry . Subunitvaccines . MAP expvaccines . Heparinvaccines . This anl domain  and the nfection , 14. Fibnfection . FAP indin vitro . Despite systems . P. pastble form , 19. Strble form . Based opPICK9KHBHA-FAP construction and cloning procedure: Sequences registered at NCBI with accession numbers of KF021287 for FAP-P gene (990 bp) and KC920678 for HBHA gene (618 bp) were analyzed. To design fusion gene, an encoded protein of FAP-P was assessed by CLC software (main workbench 5) as to determine high antigenic part among the full-length sequence of the gene. For better folding of HBHA, rigid linkers group was chosen to be introduced between two genes [wo genes .Pichia pastoris, using codon optimization tool (https://eu.idtdna.com/CodonOpt). The Pichia pastoris codon usage table was adapted from a codon usage database (http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=4922). Full-length codon optimized HBHA-FAP gene in pPICK9K yeast transfer vector was synthesized by Biomatik company (Canada), so the gene fragment was located between EcoRI and NotI cut sites under AOX1 promoter in fusion with S. cervecea alpha secretory signal at N-terminus. Pichia pastoris strain GS115  was selected as the expression host and was grown at 28°C in YPD agar medium . Codon optimization of nucleotide sequence of the fusion gene was done for increasing the expression level of the desired protein in E. coli DH5α was grown at 37°C in Luria-Bertani medium . The synthetic pPICK9KHBHA-FAP was transformed into E.coli DH5α strain via chemical transformation method. Recombinant transformants were selected by culturing in LB medium containing 50μg/ml ampicillin. Digestion of plasmid DNA using EcoRI and NotI enzymes  was done for confirmation of positive bacterial transformants.For cloning procedures, Expression analysis and optimization of HBHA-FAP expression: The recombinant plasmid pPICK9KHBHA-FAP was linearized by digesting it with SacI enzyme  in order to integrate the transgene at His4 locus in the P. pastoris genome and generating HIS+, Mut+ transformants. Ten micrograms of the linear DNA were used to be transformed into fresh electrocompetent P. pastoris cells. Electroporation was performed using Bio-Rad Gene Pulsar Xcell™ electroporation system  with a voltage of 2000V, 25μF capacitance and 200Ω resistance. The electroporated cells were immediately diluted in 1 mL of ice-cold 1 M sorbitol. 200-µL aliquots were spread on MD plates  and incubated at 30°C for 3 days. For negative control, pPIC9K without insert, linearized with SacI, was also transformed similarly. The colonies obtained were streaked on fresh MD plates.P. pastoris, along with the negative control (P. pastoris transformed with pPICK9K without insert), was inoculated separately into 25 ml of Buffered Minimal Glycerol medium-BMGY , 4×10-5% biotin, 1% glycerol) taken in 250 ml conical flask, and was incubated at 30°C in a shaker incubator at 250 rpm until the A600nm of the culture reached 2-6. The cells were harvested by centrifugation at 3,000 × g for 15 min at room temperature and the cell pellets were resuspended in the required volume of Buffered Minimal Methanol medium-BMMY (BMGY that contains 1% methanol instead of glycerol) to get an A600nm of 1 in 1000 ml conical flasks. Three methanol concentrations  for induction of expression were tried in both media in order to determine the optimum concentration of methanol. Incubation was continued in the orbital shaker incubator  for five days. To maintain the induction, methanol was added to culture the media (0.5% v/v) once in every 24 hours after collecting culture supernatants.The glycerol stocks of the positive transformants of The culture supernatants under optimal expression conditions were collected. The secreted protein was removed by pelleting the cells, and an equal volume of 20% trichloroacetic acid was added to the supernatant. The mixture was incubated overnight at -20°C. For harvesting the protein precipitate, the mixture was centrifuged at 12,000g for 30 min, and washing of the pellet was done with acetone. Eventually, the resulting protein was resuspended in phosphate-buffered saline (PBS) for next steps.SDS-PAGE and Western blot analysis of fusion protein: The concentrated proteins were analyzed by running them on 12% polyacrylamide gel electrophoresis under denaturing condition. Then, the gels were stained with Coomassie Brilliant Blue R-250 . Western blotting was performed for identification of the histidine tag expressed of the recombinant fusion protein. Hence, the separated proteins by SDS-PAGE were electrotransferred onto a nitrocellulose membrane and incubated with a 1:1000 dilution of monoclonal anti-polyhistidine-peroxidase . The color developing was done using H2O2/DAB substrate/ chromogen .FAP-P from amino acid 125 to 205 (240 bp of the gene) and the entire gene of HBHA were selected. For the evaluation of the secondary structure and selection of appropriate linker, amino acid sequences of HBHA and fusion protein were assessed before and after the fusion with a linker. At least (Pro Glu)7 were used as linker. Three-dimensional structure of HBHA and the chimeric protein of fusion gene were obtained as PDB and the similarity index between HBHA domain of chimeric protein and its native structure was obtained by TM-score measurement  in the pPICK9KHBHA-FAP construct was employed, resulting in the release of the matured, fully processed HBHA-FAP protein. Some of the His+ Mut+ colonies that were found positive through selecting media were subcultured and definitely positive samples were stored in -80°C with glycerol. Three colonies were selected for inducing the expression of target gene. Expression of HBHA-FAP recombinant protein, with a size of ~35 KDa, was detected in two clones after 72 hr of induction. The best expression level was obtained 120 hr post-induction with 0.5% methanol concentration (Data not shown). With the above optimized expression conditions, properly folded -FAP was secreted into the medium as a soluble protein. Western blotting using HRP-anti-polyhistidine  confirmed the protein expression. Proper positive signals were obtained only in culture supernatants of positive yeast transformants, not with pPICK9K negative control  , antigengenicity . It has nfection , 14. This a host .M. tuberculosis and M. bovis and reported to be a dominant secreted antigen [M. avium that presented FAP peptide to T cells contribute to the development of protective immunity to this antigen [in silico analysis and the reports on the molecular size of HBHA in previous studies [FAP-P interacts with FN for attachment and internalization of MAP by epithelial cells, and induces Th1 polarization and IFN-γ production by T cells in vitro . FAP has antigen , 33. Als antigen . Previou antigen , 35. In  antigen . Using w studies , 29.in silico analysis and choosing P. pastoris as a host, the desired fusion protein had a similar 3D structure to that of HBHA with its native form and post-translational methylation in C-terminal. Hence, the uptake of the purified fusion protein is done by M cells because of HBHA being an adhesion, and cell-mediated immunity is induced against both antigens. Checking the potential of the designed fusion protein in activation of cell-mediated immunity in animal models is being done in our laboratory.To date, researchers have focused on cell-mediated immune responses to identify candidate vaccines and in this investigation, a new fusion protein consisting of HBHA and high antigenic region of FAP-P was introduced. Based on


Microcystis flos-aquae and colonial Anabaena flos-aquae. The effect of small-scale turbulence examined varied from 0 to 8.01×10−2 m2s-3, covering the range of turbulence intensities experienced by cyanobacteria in the field. The results of photosynthesis activity and the cellular chlorophyll a in both strains did not change significantly among the turbulence levels, indicating that the potential indirect effects of a light regime under the gradient of turbulent mixing could be ignored. However, the experiments demonstrated that small-scale turbulence significantly modulated algal nutrient uptake and growth in comparison to the stagnant control. Cellular N and C of the two stains showed approximately the same responses, resulting in a similar pattern of C/N ratios. Moreover, the change in the phosphate uptake rate was similar to that of growth in two strains, which implied that growth characteristic responses to turbulence may be dependent on the P strategy, which was correlated with accumulation of polyphosphate. Additionally, our results also showed the filament length of A. flos-aquae decreased in response to high turbulence, which could favor enhancement of the nutrient uptake. These findings suggested that both M. flos-aquae and A. flos-aquae adjust their growth rates in response to turbulence levels in the ways of asynchronous cellular stoichiometry of C, N, and P, especially the phosphorus strategy, to improve the nutrient application efficiency. The fact that adaptation strategies of cyanobacteria diversely to turbulence depending on their physiological conditions presents a good example to understand the direct cause—effect relationship between hydrodynamic forces and algae.The main goal of the present work is to test the hypothesis that small-scale turbulence affected physiological activities and the morphology of cyanobacteria in high turbulence environments. Using quantified turbulence in a stirring device, we conducted one set of experiments on cultures of two strains of cyanobacteria with different phenotypes; i.e., unicellular  Accelerated eutrophication of freshwater bodies often leads to the excessive proliferation of phytoplankton, of which cyanobacteria tend to predominate. Rapid growth of cyanobacteria often leads to blooms that can have serious adverse effects on aquatic communities in lakes throughout the world . CyanobaTurbulence mixing has been widely accepted as a source of external energy to the planktonic environment, such as regulating light accessibility in the water column , bringin2-fixation [Cyanobacteria have constantly been subjected to a complex field environment imposed by these small-scale turbulent conditions. Survival of different cyanobacteria species in the mixed water column depends on the combined effects of varying environmental factors . Nutrienfixation , light tfixation , and efffixation , 24, cyafixation , 26. NevOscillatoria appears to be more shear tolerant than the heterocystous Anabaena [Many studies have reported that heterogeneity in response to turbulence is often observed among phytoplankton taxa , and cerAnabaena , 29. FurAnabaena –30. SomeAnabaena .Microcystis flos-aquae and colonial Anabaena flos-aquae. To our knowledge, few experiments have been performed employing quantified turbulence levels to evaluate the effect of small-scale turbulence on the physiology of cyanobacteria. Thus we developed a model to quantify turbulence exposure in the experiment and systematically investigate the change in photosynthesis and total cellular C, N and P of the two stains, and filament length of A. flos-aquae under different turbulent dissipation rates. The data from the study were analyzed to illustrate the mechanisms of cyanobacteria with different morphology adaptation strategies under small-scale turbulence mixing.Hard evidence of a direct cause—effect relationships between turbulence and algae is not easy to find , 31. In M. flos-aquae FACHB1028 and colonial A. flos-aquae FACHB245 were obtained from the Freshwater Algae Culture Collection of the Institute of Hydrobiology . Routine cultures of the two strains were maintained in BG11 medium with a phosphate concentration of 2 mg/L [-2 s-1 with a 12:12 light:dark cycle at a temperature of 25±1°C, and they were manually shaken 3 times every day during incubation.Unicellular f 2 mg/L  under a In the experiments, cells at their exponential growth phase were served with P free BG11 medium for a period of 10 days before the growing experiment. After that, they were inoculated into 1 L glass beakers with 2 mg/L phosphate concentration BG11 medium and exposed to different agitation intensities. A 1-L beaker was used as the reactor in the experiment. A self-designed impeller was located at the center of the beaker. Geometric dimensions are shown in −4, which was a factor by which the initial mass flow residual reduced as the simulation progressed. The simulations were carried out on the 6 nodes, 96 processor SUGON cluster. A commercial 2D particle image velocimetry (PIV) system , including a laser , a PIV camera , a synchronizer and Vidpiv 4.7 software, was employed to validate the model. The setup and data processing were the same as described earlier [A two-phase flow Computational Fluid Dynamics (CFD) model was used to simulate hydrodynamics in the reactor under different rotation speed . The who earlier . Detail A. flos-aquae colonies were treated with ultrasonication for 30 s or 60 s at 20 kHz and 100 W  to obtain single cells. The growth rate was measured during the exponential growth phase according to the exponential growth equation: μ = (lnBt2-lnBt1)/Δt [2 and Bt1 represent the cell concentration at the end (t2) and beginning (t1) of the log phase, respectively, and Δt = t2-t1. For Chla measurements, the samples were centrifuged at 6,000 rpm for 15 min, and cell pellets were extracted with 80% acetone/20%water.Two strains were batch cultured under different agitation intensities (0 rpm to 500 rpm) for two weeks and were sampled every 2 days for cell counting under a microscope  using the appropriate magnification and a hemocytometer. Prior to cell counting, nBt1)/Δt , where μIn vivo chlorophyll fluorescence was measured with a phytoplankton analyzer . The samples were dark-adapted for at least 15 min before measuring the fluorescence parameters . The electron transport rate of PSII (ETR) was calculated according to [Fm- Ft) /Fm)×0.84×0.5×PAR (m-2s-1)). The maximum effective quantum yield of PSII was calculated as Fv/Fm = (Fm-F0)/Fm [Fv is the difference between Fm and F0, which are the maximum and minimum fluorescence values of the dark adapted stage of PSII.rding to . Relativm-F0)/Fm . Fv is tTo measure the carbon, nitrogen and phosphorus contents of the two strains, culture suspensions were taken from each sample, filtered through GF/F filters  and dried at 65°C for 24 h. Each filter was split for analysis of C, N, and total cellular phosphorus (TCP). C and N concentrations were measured using a Vario El cube elemental analyzer . The filtrates were used for measuring soluble reactive phosphorus (SRP). The filtered extracts were diluted with distilled water, and TCP was determined by digestion , 39 foll(W0-Wt) · V · N0-1· t-1, where W0 is the initial concentration of phosphate, Wt is the concentration of the phosphate after t hours, N0 is the initial level of cell density and V is the volume of culture medium.Phosphate uptake rates were calculated by the change in phosphate concentration in the culture media during 72 h batch cultures at the same culture conditions as mentioned above. Phosphate levels were determined using ascorbic acid-molybdenum blue spectrophotometric methods . The uptAnabaena filaments were examined using an Olympus BX 53 light microscope with a digital camera . The sizes of the filaments were measured in microphotographs using image analysis Olympus DP-Soft. One hundred filaments were chosen at random from each sample to capture the images, and then the surface area (S), width (d), and length (l) of individual filaments were obtained using Olympus DP-Soft, which was calibrated separately for each magnification.P < 0.05.The data in this study are presented as the mean ± standard deviation (SD). Significant differences between controls and treated samples were determined with an ANOVA. All statistical analyses were carried out with Origin 8.0 . Differences were considered to be significant at M. flos-aquae and colonial A. flos-aquae at different turbulent dissipation rates are shown in M. flos-aquae, the growth rate increased with the elevation of the turbulent dissipation rate. At a turbulent dissipation rate of 1.51×10−3 m2s-3, the growth rate of M. flos-aquae was approximately 3.4 times than that in the stagnation condition. When the turbulent dissipation rate was above 5.06×10−2 m2s-3, the growth rate of M. flos-aquae remained relatively unchanged . On the other hand, the growth rate of A. flos-aquae increased dramatically  at lower turbulent dissipation rates and reached the max at 2.26×10−2 m2s-3, while the growth rate decreased with increases in the turbulence intensity at higher levels. Overall, the growth rates of A. flos-aquae at all levels of turbulence intensity were lower than that of M. flos-aquae.Specific growth rates of unicellular max) and the maximum quantum yield (Fv/Fm) of M. flos-aquae and A. flos-aquae showed considerable differences (M. flos-aquae were significantly higher than in the control (non-turbulent) treatment . Among all the turbulent mixing treatments, markedly low values of Fv/Fm of M. flos-aquae were found in cells grown at a turbulence intensity of 1.51×10−3 m2s-3. The values of ETRmax and Fv/Fm were not found to be significantly different among levels of turbulence intensities from 2.26×10−2 to 8.01×10−2 m2s-3 . Coincident with the elevation in the ETRmax of M. flos-aquae, the changes in the ETRmax of A. flos-aquae showed similar trends to that of M. flos-aquae . However, it appears that there was no significant difference in the Fv/Fm of A. flos-aquae between all the turbulent treatments and the controls . The cellular Chla showed a similar pattern to the Fv/Fm ratios, although the cellular Chla was relatively higher at higher turbulent dissipation rates, probably due to the reduction in cell density at the highest turbulent dissipation rates  were significantly lower than that in the turbulent dissipation rates of 1.51×10−3 and 6.63×10−3 m2s-3 at the end of the test . However, the SRP concentration in the A. flos-aquae culture with a turbulence intensity of 2.26×10−2 m2s-3 was the greatest at the end of the test, whereas no significant differences were observed among the other treatment cultures . The phosphate uptake rate was analyzed as a function of the external phosphate concentration. It showed similar trends to that of SRP .The time course of SRP in the culture medium and phosphate uptake rate of t of SRP . Under tM. flos-aquae, cellular C was the highest under the turbulent dissipation rate of 5.06×10−2 m2s-3, with an increase of 11% compared with the control treatment. However, the cellular N concentration was significantly decreased with increasing turbulence intensity, except under the turbulent dissipation rate of 1.51×10−3 m2s-3. Furthermore, as dissipation rates increased, there was also an increase in cellular P, and the maximum value was observed under the turbulent dissipation rate of 2.26×10−2 m2s-3, which was 2.0-fold compared with that at the stagnation condition. Additionally, the changes in cellular PolyP showed similar trends to that of cellular P .To further explore the cellular stoichiometry changes, cellular C, N and P in response to different turbulent dissipation rates were analyzed . There wllular P . In A. fM. flos-aquae and A. flos-aquae are also shown in M. flos-aquae and A. flos-aquae in response to small-scale turbulence. There was an increase in the C/N ratio and a decrease in the N/P ratio in M. flos-aquae at the dissipation rate of 6.63×10−3 m2s-3 and above, compared with the controls. Variations in the C/N ratio in A. flos-aquae were similar to that in M. flos-aquae. The maximum C/N ratio was recorded at the turbulent dissipation rate of 2.26×10−2 m2/s3. However, the N/P ratio in A. flos-aquae decreased only at the turbulent dissipation rates of 6.63×10−3 and 2.26×10−2 m2s-3, compared with the control treatment, which was due to the increase in cellular P.Changes in molar ratios of C/N and N/P of A. flos-aquae varied with different turbulent dissipation rates  was the lowest. The frequencies of 100–200 μm lengths decreased at the turbulent dissipation rates of 1.51×10−3 and 6.63×10−3 m2s-3, while for the high turbulence intensities of 8.01×10−2 m2s-3, the frequencies of large filaments (>200 μm) also decreased significantly.The filament length of on rates . It was −4 to 10−2 m2s-3. On the Kolmogorov microscale, it was in the higher range of turbulences in natural aquatic ecosystems. However, it covered the range of turbulence intensities experienced by cyanobacteria in the field [Turbulence is ubiquitous in aquatic systems and thus can potentially influence a wide scope of phytoplankton and eco-physiological processes. It is still often referred to as one of the unsolved problems in field studies due to the interactions with other variables, such as light penetration and supply of nutrients, and their consequences for organism physiology and behaviors . Some ofMicrocystis and Anabaena to turbulence intensities were quite different as inferred by growth. The turbulent regimes of 1.51×10−3 to 2.26×10−2 m2s-3 favored the growth of the two strains. However, in regard to the higher dissipation rate of 8.01×10−2 m2s-3, turbulence had a negative impact on the growth rate of A. flos-aquae but did not affect the growth of M. flos-aquae, suggesting that unicellular M. flos-aquae was better adapted to growing under high turbulent dissipation rates.In most studies described in the literature to date, the effect of turbulence on phytoplankton was determined by changes in growth and the viability of the organism , 47. In Fv/Fm and ETRmax are considered as the sensitive photosynthetic parameters to environmental stress [Fv/Fm and ETRmax in the exponential phase support the hypothesis that the photosynthetic activity of A. flos-aquae significantly increases when compared with stagnant conditions but might not change in contrast to the unimodal response of a growth rate to a gradient of turbulent mixing. Similar results were also discussed by [Chla content in both M. flos-aquae and A. flos-aquae increased with turbulence even under a higher level, indicating that the photosynthetic apparatus of algae showed little or no disruption in response to turbulence. Therefore, the potential indirect effects of a light regime under the gradient of turbulent mixing, e.g., the shading effect due to an increase in the population in the reactors, could be ignored.The photosynthetic system is a good indicator system because it is very sensitive to any changes in living conditions . Fv/Fm al stress . At the ussed by , indicatA great majority of studies reported that turbulence may have beneficial effects on phytoplankton due to an increase in the diffusive transport of nutrients toward the cell surface and, hence, an increase the nutrient uptake rate , 51. UndM. flos-aquae, the concentration of PolyP in A. flos-aquae at dissipation rates of 2.26×10−2 to 5.06×10−2 m2s-3 were much higher than those with a dissipation rate of 1.51×10−3 m2s-3, indicating that the reallocation of cellular P in A. flos-aquae could be a response to the changed turbulent conditions, and the filament integrity in A. flos-aquae was more sensitive to turbulence than in unicellular M. flos-aquae. Furthermore, our results also showed that the filament length of A. flos-aquae decreased in response to turbulence. It seemed that turbulence negatively affects cyanobacterial morphology prior to physiological activity. We stress that this may be related to the nutrient acquisition strategies, which was depended on algal size. Theoretical predictions regarding the thickness of external diffusion boundary layer is equal to the equivalent spherical diameters of the organism [A. flos-aquae at higher turbulent dissipation rates could favor the resource gain. Our findings support what had been suggested by [Formation and storage of PolyP is commonly regarded as a nutritional strategy of microalgae when encountering environmental stress . Accordiorganism . Thus, lorganism , we infeested by , such thM. flos-aquae and A. flos-aquae adjust their growth rates in response to turbulence levels in the ways of asynchronous cellular stoichiometry of C, N, and P, especially the phosphorus strategy, to improve the nutrient application efficiency. Additionally, the colonial A. flos-aquae could change the effective filament length to enhance the nutrient uptake under high turbulent conditions. Again, these evidences supported the hypothesis that hydrodynamic regimes may impact eco-physiology of cyanobacteria to form certain adaptive strategies, which should be carefully considered in elucidating mechanisms of cyanobacteria blooms. Clearly, future studies are warranted to assess the effects of turbulence on nutrient flux of cells including different morphological cyanobacteria species.A gradient of increasing levels of dissipation rates is useful when attempting to investigate the shape and character of potential relationships between small-scale turbulence, nutrient conditions and planktonic organisms . Small-sS1 File(DOCX)Click here for additional data file.


Antrodia camphorata is a fungus that is endemic to Taiwan, and its fruiting body has been used as a folk medicine for the prevention or treatment of diverse diseases. The present study is aimed at investigating the antimelanogenesis and antioxidation effect of the ethanolic extract of Antrodia camphorata fruiting body (EE-AC), as well as its antiproliferation effects in B16-F0 melanoma cells. Regarding antimelanogenic effects, EE-AC had effective cupric ions reducing capacity and expressed more potent inhibitory effect than kojic acid on mushroom tyrosinase activity. Moreover, EE-AC significantly inhibited cellular tyrosinase activity and the melanin content in B16-F0 cells at 12.5 μg/mL concentration without cell toxicities. Regarding antioxidant effects, EE-AC exhibited potent DPPH radical- and SOD-like-scavenging activities. Regarding antiproliferative effects, EE-AC exhibited a selective cytotoxic effect and markedly inhibited the migration ability of B16-F0 cells. EE-AC increased the population of B16-F0 cells at sub-G1 phase of the cell cycle. EE-AC also caused the increase of early apoptotic cells and chromatin condensation, which indicated the apoptotic effects in B16-F0 cells. We demonstrated that EE-AC possessed antimelanogenic, antioxidant and anti-skin cancer actions. The results would contribute to the development and application of cosmetics, healthy food and pharmaceuticals. Antrodia camphorata (synonym Antrodia cinnamomea) is a highly valued mushroom that is endemic to Taiwan. It is rare and cannot be cultivated because it grows only on the inner heartwood wall of the endemic evergreen Cinnamonum kanehirai [A. camphorata has been traditionally used as a medicine to treat food intoxication and liver disease by the aboriginal Taiwanese [A. camphorata crude extract has been proven to possess numerous healthy characteristics and functional metabolites [A. camphorata are yet to be identified.anehirai . The fruaiwanese . A. campabolites . HoweverMelanoma is a highly aggressive and invasive skin cancer, and occurs most commonly among the Caucasian. It is usually caused by skin cell damage resulting from exposure to ultraviolet light from the sun. Melanogenesis is a physiological process that results in the synthesis of melanin pigments, and tyrosinase is a key enzyme for melanin biosynthesis . MelaninA. camphorata mycelia have been found to be free radical scavengers in possessing antioxidant activity [A. camphorata fruiting bodies. The anti-tumor physiological effects of A. camphorata fruiting bodies have been reported in various cancers in vitro and in vivo [A. camphorata could inhibit melanoma proliferation and progression [A. camphorata exert anticancer effects on human pancreatic cancer cells [A. camphorata fruiting body to kill melanoma cells and the main mechanisms that underlie its anticancer effect need to be further investigated.Regarding antioxidant effects, the extracts from activity . However in vivo –10. Recegression . We prever cells . HoweverA. camphorata are not fully investigated and its other effects have not been clarified, as part of our continued search for novel bioactive natural food, we investigated the antimelanogenesis, antioxidation effect of the ethanolic extract of A. camphorata fruiting body (EE-AC), as well as its antiproliferation effects in B16-F0 mouse melanoma cells. This study is the first to explore new applications of A. camphorata, which provides valuable information about the development of potential depigmenting agents such as skin-whitening cosmetics and a multifunctional healthy food for skin cancer prevention.Since the traditional medicinal uses of Annexin V-FITC apoptosis detection kits were purchased from Strong Biotech . Cisplatin, MTT, PI and other chemicals which not specified above were purchased from Sigma Chemical Co. .A. camphorata (grown on the root of wild C. kanehirai Hay) was provided by Sky Tree Biotechnology Co., Ltd ; it was collected from mountain areas in southern Taiwan. Preparation of the EE-AC was performed as previously described [g for 30 min. Later, a rotary evaporator under vacuum was used for the lyophilization process of the ethanol extracts.The fruiting body of escribed . Brieflyg for 15 min, dried and dissolved with distilled water. A reading of 490 nm in a spectrophotometer was recorded.The concentration of polysaccharides was measured according to the procedures of a previously published method by the phenol-sulfuric acid assay using glucose as a standard . The extMeasurement of the total (1→3)-β-D-glucan concentration was performed according to the procedures of a previously published method by the aniline blue assay . BrieflyA. camphorata fruiting body was dissolved in 10 mL of 100% methanol and then filtered through a 0.22 μm membrane to prepare the sample solution. The 10 μL sample solution was analyzed using HPLC . The detection of the absorbance using a photodiode array detector at 275 nm was carried out. The model of the analytical column was Intertsil ODS-3 C-18 C18 column, 25 cm × 4.6 mm i.d., 5 μm. The mobile solution eluded, containing of 0.2% formic acid and 100% acetonitrile at a flow rate of 1.0 mL per min was performed. A colorimetric method was used to determine the total triterpenoid content of A. camphorata fruiting bodies. Briefly, under reflux in 5 mL of 95% ethanol for 30 min, the extraction of 0.5 g sample was gained. The extraction was then filtered and centrifuged at 3000×g for 10 min. One mL of the diluted solution (1/10) was dried, and 0.2 mL of 5% (w/v) vanillin–glacial acetic acid and 0.08 mL of perchloric acid were added sequentially after the extraction was done. The mixture was shaken in a 60°C water bath for 20 min. After being added to 3.72 mL of glacial acetic acid against a standard oleanolic acid curve, a 550 nm absorbance  was used to read the concentration of total triterpenoids.Qualitative analysis of total triterpenoids was performed according to the procedures of a previously published method . BrieflyThe concentration of total phenolic compounds was measured according to the procedures of a previously published method . Regardi2, and 4% NaOH (2.0 mL) were well mixed and kept at room temperature for 15 min. Next, a spectrophotometer at 415 nm was used to measure the absorbance. Lastly, calculation of the total flavonoids content was determined based on an established standard curve of rutin.The concentration of total flavonoids was measured according to the procedures of a previously published method . For mea2+ reducing power was performed according to a previously published method [4 aqueous solution (0.4 mM) mixed vigorously, and incubated for 10 min. Then, a 0.4 mL of bathocuprinedisulfonic acid (4 mM) was added to the mixture. Absorbance against a buffer blank was measured at 483 nm after 10 min.Measurement of Cud method . BrieflyMeasurement of mushroom tyrosianse inhibition was performed as described previously with slight modifications . A volum™) and human embryonic kidney cells  were cultured with DMEM supplemented with 10% fetal bovine serum with the mixture of 100 IU/mL penicillin and 100 μg/mL streptomycin at 37°C in a 5% CO2 humidified incubator.B16-F0  were plated in each well of a 24-well plate and incubated for 48 h. Then, the cells were lysed with 1% Triton-X by freeze-thawing. The lysate was centrifuged at 16000×g for 20 min. Finally, 100 μL of each supernatant was added to 100 μL of 2 mM L-DOPA and absorbance was measured at 492 nm in a microplate reader.Measurement of intracellular tyrosinase activity was performed according to a previously published method with slight modifications . In brie4 cells/0.5 mL of medium containing EE-AC) were plated in each well of a 24-well plate and incubated for 48 h. Then, the cells were dissolved in 100 μL of 1N NaOH after twice washing with PBS and boiled for 30 min to solubilize the melanin. The lysate was centrifuged at 16000×g for 20 min. Then, the absorbance at 405 nm of the supernatant was measured in a microplate reader.Cells . After the reaction was carried out at room temperature for 30 min, the free-radical-scavenging activity of the sample was quantified by the decolorization of DPPH at 517 nm.One milliliter of 120 μM PMS, 1mL of 936 μM NADH and 1 mL of 300 μM NBT were added to 1.0 mL sample solution sequentially. After the reaction was carried out at room temperature for 5 min, the superoxide anion-scavenging activity of the sample was quantified by the decolorization of SOD-like at 560 nm.3 cell) were plated in each well of a 96-well plate. After cells were exposed to various concentrations of EE-AC for 48 h, a 50 μL MTT solution (5 mg/mL in stock solution) was added to each well. After the cells were incubated at 37°C for 2 h, formazan crystals were formed and dissolved with DMSO (100 μL). Next, an ELISA reader was used to read the absorbance at 570 nm.Cell viability assay was performed as previously described . BrieflyWound healing assay was performed as previously described . BrieflyCell cycle analysis was performed as previously described . BrieflyAccording to the manufacturer’s operation manual, the cells were stained with Annexin V-FITC and PI. A FACScan flow cytometer was used to analyze each sample of at least 10,000 cells after the cells were incubated in the dark at 25°C for 15 min.Examination of the nuclear morphology was performed as previously described . Brieflyt-test. A P value of less than 0.05 was considered as statistically significant.Data are presented as means ± SD and analyzed using Student's Antrodia camphorata fruiting body. The yields in weight percentage of residues, referring to the weight of dry material extracted, were 3.59% of EE-AC (w/w). The EE-AC contains certain levels of bioactive compositions, including total polysaccharides, 1,3-β-D-glucan, triterpenoids, polyphenols and flavonoid. The total polysaccharide content in EE-AC was 202.35 ± 15.24 mg/g. The total 1,3-β-D-glucan content in EE-AC was 81.34 ± 3.89 mg/g. The total triterpenoid content in the A. camphorata fruiting body was determined using oleanolic acid as the reference standard to be 5.62 ± 0.64%. The total polyphenol content of EE-AC was 78.56 ± 6.86 mg of gallic acid equivalent/g of extract. The flavonoid production of EE-AC was 11.52 ± 1.85 mg of rutin equivalent/g of extract . The IC50 value for EE-AC on the mushroom tyrosinase activity was found as 1.25 mg/mL  of cisplatin at 48 h was 10 μM. In the tested groups, EE-AC was found to be toxic to B16-F0 cells with IC50 values of 50 μg/mL, and posed no significant toxicity to human normal HEK-293 cells at the same concentration. It is worth noting that EE-AC was proved to have good cellular inhibition of tyrosinase activity and melanin production at 12.5 μg/mL, and had no cytotoxicity effect to human normal cells.We examined the effect of EE-AC on the viability of B16-F0 cells using MTT assay. As shown in Melanoma is a highly malignant tumor with a high metastatic rate. Hence, wound healing assay was performed to evaluate cancer cell migration ability. We compared EE-AC with the negative control group (0.1% DMSO) and the positive control group (10 μM cisplatin). The results demonstrated that EE-AC significantly suppressed the migration of B16-F0 cells in a time-dependent manner, and its inhibitory effect was even more significant than that of cisplatin, a common chemotherapeutic agent for solid malignancies .The decreased cell viability could be the result of the inhibition of growth and/or the induction of apoptosis. We therefore investigated whether EE-AC could induce cell cycle arrest and apoptosis in melanoma cells. To examine the effect of EE-AC on cell cycle regulation in B16-F0 cells, the ploidy state of cells was monitored by flow cytometry after PI staining nuclei. As shown in To prove that EE-AC inhibited B16-F0 cell proliferation by induction of apoptosis, the percentage of apoptotic cells was examined on phosphatidylserine externalization in B16-F0 cells and flow cytometric analysis. As shown in In vitro testing revealed that EE-AC possessed great efficacy of tyrosinase inhibitory activity as evident by its Cu2+ reducing ability. Therefore, EE-AC is also a good chelator of copper. One of the interesting findings of our study is that although AC-EE is not a pure compound, it exerts comparable antimelanogenesis activity as kojic acid, which suggests that it should have the potential to be developed into a whitening agent. Because safety is a major concern when developing a potential whitening agent, cell viability assay was performed in B16-F0 melanoma cells and HEK-293 human normal cells to assess the cell safety of EE-AC. We found that EE-AC at a concentration of 12.5 μg/mL has a strong antimelanogenic effect on B16-F0 cells but does not show any cytotoxic effect on B16-F0 and HEK-293 cells. This is the first report on the antimelanogenesis effect of A. camphorata, and the results showed that EE-AC may be superior to kojic acid as a whitening agent because of its natural source, predominant bioactive compounds and safety. Further investigations are required to verify the antimelanogenic effect of EE-AC in an animal model.We found a new natural resource, EE-AC that shows high inhibitory activities against melanogenesis. Kojic acid has been shown to inhibit tyrosinase from various sources including fungi, plants, and animal cells; its depigmenting action is attributed to an ability to chelate copper at the active site of tyrosinase that may lead to the loss of tyrosinase activity . In vitrA. camphorata fruiting body into healthy food supplements for effective chemopreventive treatment for skin cancer. Nevertheless, further research on the effectiveness of EE-AC treatment in melanoma cells is still necessary. It has been reported that some fungal and plant extracts, which are rich in bioactive metabolites, are beneficial for melanoma prevention and therapy [Melanoma is a highly aggressive skin cancer due to its metastasizing potential and resistance to conventional cancer therapies. Cell migration is essential for cancer cell invasion and metastasis. In this study, the migration rate of B16-F0 cells was markedly inhibited by exposure to EE-AC, and this inhibitory migration effect was more significant than that of cisplatin, a common chemotherapy drug for various types of cancers. We also demonstrated that the decrease in cell viability by EE-AC was due to the induction of cells with a sub-diploid DNA content, and apoptosis in B16-F0 cells. The proapoptosis effect induced by EE-AC was evidenced as the increase of apoptotic cells by FITC-conjugated annexin V and chromatin condensation measurement, indicating that EE-AC-induced cytotoxicity was involved in the apoptosis mechanism. In addition, this proapoptosis effect of EE-AC was more significant than that of cisplatin. The inhibition of migration and viability results imply that EE-AC is a potential anticancer agent for skin cancer. The results may provide new insight and deepen the understanding of the chemopreventive properties of EE-AC. Moreover, these results offer the possibility of developing the  therapy –20. In tA. camphorata [A. camphorata have been found to be an edible free radical scavenger in preventing oxidative damage in living organism [A. camphorata are also related to anticancer properties [A. camphorata [A. camphorata also possessed potential applications in antioxidant activity and antiproliferative properties [A. camphorata fruiting body are predominantly polysaccharides, 1,3-β-D-glucan, triterpenoids, polyphenols and flavonoid; this suggests that these five functional components of A. camphorata should contribute to the regulation of melanoma cell melanogenesis, oxidation and growth. However, the specific effect of each bioactive ingredient of A. camphorata need further study.In the present study, the results demonstrated that like vitamin C, EE-AC also possesses potent DPPH radical and SOD-like scavenging activities, which verifies previously reported data revealing antioxidant activity and showing as a free radical scavenger of mphorata . Polysacorganism ,22. Receorganism . Recentloperties . In addimphorata ,24,12. Poperties ,25. TherA. camphorata fruiting body could be an effective substance in skin-whitening cosmetics and healthy food supplements, as well as a chemopreventive agent against human skin cancer.In sum, the present study demonstrated, for the first time, the potential effects of EE-AC have antimelanogenetic, antioxidant and antiproliferative activities in melanoma cells. These results suggested that the